Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples

Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Review, Food and Drug Administration, Bethesda, MD 20892, USA.
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 06/2007; 356(4):1017-23. DOI: 10.1016/j.bbrc.2007.03.087
Source: PubMed


Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.

Download full-text


Available from: Angamuthu Selvapandiyan, May 09, 2014
47 Reads
  • Source
    • "Simple and inexpensive molecular assays based on dipstick and zipper technology have also been described.77,78 The Cepheid GeneXpert System (Sunnyvale, CA, USA) is a single-use sample processing cartridge system with an integrated multicolor real-time PCR capacity.79 Microarrays have also been incorporated with nucleic acid probes and peptides to detect and quantify HIV-1.80,81 Miniaturized PCR devices have been reported for microbial agent detection and identification. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Substantive and significant advances have been made in the last two decades in the characterization of human immunodeficiency virus (HIV) infections using molecular techniques. These advances include the use of real-time measurements, isothermal amplification, the inclusion of internal quality assurance protocols, device miniaturization and the automation of specimen processing. The result has been a significant increase in the availability of results to a high level of accuracy and quality. Molecular assays are currently widely used for diagnostics, antiretroviral monitoring and drug resistance characterization in developed countries. Simple and cost-effective point-of-care versions are also being vigorously developed with the eventual goal of providing timely healthcare services to patients residing in remote areas and those in resource-constrained countries. In this review, we discuss the evolution of these molecular technologies, not only in the context of the virus, but also in the context of tests focused on human genomics and transcriptomics.
    Emerging Microbes and Infections 08/2012; 1(8). DOI:10.1038/emi.2012.15 · 2.26 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Currently, there are nine known human herpesviruses and these viruses appear to have been a very common companion of humans throughout the millenia. Of human herpesviruses, herpes simplex viruses 1 and 2 (HSV-1, HSV-2), causative agents of herpes labialis and genital herpes, and varicella-zoster virus (VZV), causative agent of chicken pox, are also common causes of central nervous system (CNS) infections. In addition, human cytomegalovirus (CMV), Epstein-Barr virus (EBV) and human herpesviruses 6A, 6B, and 7 (HHV-6A, HHV-6B, HHV-7), all members of the herpesvirus family, can also be associated with encephalitis and meningitis. Accurate diagnostics and fast treatment are essential for patient recovery in CNS infections and therefore sensitive and effective diagnostic methods are needed. The aim of this thesis was to develop new potential detection methods for diagnosing of human herpesvirus infections, especially in immunocompetent patients, using the microarray technique. Therefore, methods based on microarrays were developed for simultaneous detection of HSV-1, HSV-2, VZV, CMV, EBV, HHV-6A, HHV-6B, and HHV-7 nucleic acids, and for HSV-1, HSV-2, VZV, and CMV antibodies from various clinical samples. The microarray methods developed showed potential for efficiently and accurately detecting human herpesvirus DNAs, especially in CNS infections, and for simultaneous detection of DNAs or antibodies for multiple different human herpesviruses from clinical samples. In fact, the microarray method revealed several previously unrecognized co-infections. The microarray methods developed were sensitive and provided rapid detection of human herpesvirus DNA, and therefore the method could be applied to routine diagnostics. The microarrays might also be considered as an economical tool for diagnosing human herpesvirus infections. Herpesvirukset ovat kulkeneet ihmisen kumppaneina vuosituhat toisensa jälkeen ja tällä hetkellä tunnetaan yhdeksän ihmiselle patogeenista herpesvirusta. Huuli- ja genitaaliherpestä aiheuttavat herpes simplex virus 1 ja 2 (HSV-1, HSV-2) sekä vesirokkoa aiheuttava varicalla-zoster virus (VZV) ovat ihmisen herpesviruksia, jotka voivat aiheuttaa myös vakavia keskushermostoperäisiä infektioita. Ihmisen herpesviruksista myös ihmisen cytomegalovirus (CMV), Epstein-Barr virus (EBV), ihmisen herpesvirukset 6 ja 7 (HHV-6, HHV-7), voidaan liittää aivokuumeeseen ja aivokalvontulehdukseen. Näiden virusten nopea ja tarkka diagnostiikka on oleellista oikean hoidon aloittamiselle sekä potilaan toipumiselle varsinkin keskushermostoperäisissä infektioissa. Taudinaiheuttajien diagnosoimiseksi tarvitaan herkkiä ja tehokkaita menetelmiä. Tämän väitöskirjan tavoitteina oli kehittää uusia mikrosirupohjaisia menetelmiä ihmisen herpesvirusten aiheuttamien infektioiden diagnostiikkaan. Kehitetyillä mikrosiruilla tunnistettiin yhtä aikaa kahdeksan eri herpesviruksen nukleiinihappoa erilaisista kliinisistä näytteistä. Toisella serologisella mikrosirulla detektoitiin HSV-1, HSV-2, VZV ja CMV:lle spesifisiä vasta-aineita. Herpesvirusten nukleiinihappojen ja vasta-aineiden tunnistamiseen kehitetyt mikrosirumenetelmät osoittautuivat soveltuviksi usean herpesviruksen samanaikaiseen diagnosointiin. Varsinkin keskushermostopohjaisten infektioiden tunnistamisessa mikrosiru osoittautui tehokkaaksi. Mikrosirujen avulla tunnistettiin myös mahdollisia useamman viruksen samanaikaisia infektioita.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: New scientific discoveries and technologies create opportunities for medical and public health advancement through development of innovative products. However, novel products and technologies bring new challenges to regulation. FDA recently established a ‘Critical Path’ research initiative to modernize regulatory science concepts and tools to meet the challenges of the 21st century. Central to this initiative is the concept that regulatory science is distinct from the ‘discovery’ science that generates ideas for development of new drugs, biologics, or medical devices. In this article, the authors discuss the concepts of FDA ‘Critical Path’ research and review examples of such research performed in the Office of Blood Research and Review within the Center for Biologics Research and Evaluation at FDA to illustrate how the ‘Critical Path’ research is being used to address opportunities and challenges impacting blood and blood products.Section editor:Janet Woodcock – Food and Drug Administration, Rockville, MD, USA
    Drug Discovery Today Technologies 06/2007; 4(2-4):51-54. DOI:10.1016/j.ddtec.2007.10.011
Show more